Substrate Peptidomimetic Inhibitors of P. falciparum Plasmepsin X with Potent Antimalarial Activity

Abstract Plasmepsin X (PMX) is an aspartyl protease that processes proteins essential for Plasmodium parasites to invade and egress from host erythrocytes during the symptomatic asexual stage of malaria. PMX substrates possess a conserved cleavage region denoted by the consensus motif, SFhE (h=hydrophobic amino acid). Peptidomimetics reflecting the P3‐P1 positions of the consensus motif were designed and showed potent and selective inhibition of PMX. It was established that PMX prefers Phe in the P1 position, di‐substitution at the β‐carbon of the P2 moiety and a hydrophobic P3 group which was supported by modelling of the peptidomimetics in complex with PMX. The peptidomimetics were shown to arrest asexual P. falciparum parasites at the schizont stage by impairing PMX substrate processing. Overall, the peptidomimetics described will assist in further understanding PMX substrate specificity and have the potential to act as a template for future antimalarial design.


Index
Page S2 Figure S1. Overview of PMX and PMIX substrate processing.
S7 Figure S5. Correlation plot of P. falciparum viability versus PMX activity.
S8 Figure S6. Model of peptidomimetics in complex with PMX.

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References.   [1] and the invasion and egress proteins were identified. Multiple isoforms of PfEMP1, Rhoptry-associated membrane antigen and Rifin were excluded from the Table. b Consensus motif in bold text. c Leu and Ile treated as equivalent in the searches indicated. d Multiple isoforms of the protein were found. Highlighted in yellow are proteins investigated in peptide cleavage experiments by Favuzza et al. [2] Highlighted in blue are liver stage proteins.  falciparum parasites were exposed to the specified compound for 72 h at 37 °C. EC50 data represents the mean and SD for 3 independent experiments measuring LDH activity.
S7 Figure S5. The correlation of P. falciparum viability EC50 data and PMX biochemical IC50 data for tested analogues. Each data point plotted represents the IC50 versus EC50 data for an analogue tested in the biochemical PMX assay and P. falciparum viability assay, respectively. IC50 data is the mean of 3 or more replicate experiments. EC50 data is the mean of 3 or more replicate experiments.
Analogues that were inactive in the concentration range of either assay were excluded from the analysis. The analysis performed was the Pearson correlation analysis (GraphPad Prism 8.0.2) where 'r' is the correlation coefficient. Shown are the hydrogen bonds of the HEA motif of peptidomimetics with Gly233 and the catalytic Asp421 and Asp321. The model was generated using the X-ray structure of the HEA compound MR0 C 803 bound to BACE-1 (PDB: 2P83) [4] as a template for positioning the peptidomimetics and then performing minimalization to the X-ray structure of PMX (PDB: 72BC). [2]  Positions of structural moieties of peptidomimetics are labelled relative to their respective binding pockets. PMX flap amino acids Ile274 to Ile281 were excluded for clarity. The model was generated using the Xray structure of the HEA compound MR0 C 803 bound to BACE-1 (PDB: 2P83) [4] as a template for positioning the peptidomimetics and 49c and then performing minimalization to the X-ray structure of PMX (PDB: 72BC). [2] Figure S8. Replicate images of analogue 38 and 49c (2) blocking merozoite egress. Figure S9. Uncut P. falciparum processing assay western blots. Uncut western blots from the PMX processing inhibition assay (long exposure left and standard exposure middle) and uncut PMIX processing inhibition assay (standard exposure) shown in Figure 5. Positive control compound and activity in assay 49c [5] IC50 1 nM 49c [5] IC50 15,100 nM WEHI-842 [6] IC50 17 nM Aliskiren [7] IC50 2 nM Pepstatin A IC50 0.7 nM Verubecestat [8] IC50 5 nM